Cell-Free Protein Synthesis for High-Through-Put Proteomics

73169S03-I Production of proteins from molecularly cloned genes is currently a cumbersome and difficult process requiring gene cloning, vector construction, optimization of expression, and purification from the host cell source. In addition, in vivo protein expression is often compromised by cell toxicity, degradation, insolubility, and unwanted post-translation modification problems. This project will develop a new methodology for cell-free protein synthesis, which will allow production and affinity purification of 50 milligram or more quantities of active protein, using an inexpensive and highly stable wheat germ cell-free system. The method will be tested with Polymerase Change Reaction (PCR) amplified genes that are used to generate the synthetic mRNA templates for the procedure. Phase I will determine whether the cell-free synthesis system works with a variety of mRNAs; demonstrate that active protein is produced by the method; scale up to produce up to 100 mgs of protein; develop a method to affinity purify synthesized, 6xHis tagged protein by Ni-sepharose resin; determine if the method can synthesize protein from several messages at one time; develop a method to use PCR fragments to generate mRNA and subsequently protein with this system; determine whether the mRNA synthesis reaction can be done simultaneously with the protein synthesis; and estimate the cost to produce the product. Commercial Applications and Other Benefits as described by the awardee: The cell-free protein method should be commercialized in the form of kits, supplies, and services sold to research labs conducting proteomics, genomics, and protein structure/function work. These products could be directly marketed to the 50,000 laboratories throughout the world that perform molecular biology research and for whom there is a growing need for cost savings, reduced labor, reproducibility, and automation. The products will improve protein production and yield higher quality protein than any commercially available product.